A biofilm is a population of bacteria growing on a surface and surrounded by a complex extracellular polymeric substance composed of proteins, glycoproteins, glycolipids, polysaccharides, and DNA. There is increasing awareness that biofilms are involved in the majority of infectious diseases, especially those in postsurgical, trauma, and critically ill patients. These diseases include pneumonia in ventilated patients, endocarditis, burn wound infections, and infections of indwelling devices. Postsurgical, trauma, and critically ill patients are at increased risk for catheter-related infections and surgical site infections. Nosocomial infections are among the ten leading causes of death in the USA, and more than 20% of nosocomial infections are surgical site infections and a large proportion of these involve surgical suture. Vascular catheters are the most common cause of bloodstream infections and it is estimated that 250,000 catheter-associated bloodstream infections occur annually with high attributable mortality (12 to 25%) and annual costs ranging from $300 million to several billion dollars. This proposal focuses on two of the most prevalent etiologic agents of catheter-related and surgical site infections, namely Staphylococcus aureus and Enterococcus faecalis. Not only are these bacteria prevalent in biofilm-related infections, the antimicrobial resistance associated with staphylococci and enterococci continues to increase making it increasingly important to discover new strategies to prevent these infections. The force behind this proposal is our recent discovery of unusual (heretofore unrecognized), large cellular elements that appear early in biofilm formation (within 15 min) and are likely of critical importance. We have compelling preliminary evidence that these large cellular elements may not only play a pivotal role in initial attachment of the biofilm to the substratum (the surface) and may thus represent a common on-off switch in biofilm initiation, but these relatively large cells may be the putative persister cells thought to be primarily responsible for the increased antibiotic resistance of the biofilm. We have four specific aims. Aim 1 will characterize the large, newly discovered cellular elements that appear early in biofilm development. Aim 2 will compare the development and composition of S. aureus and E. faecalis biofilms cultivated in vitro on suture and on silastic catheter material, focusing on early developments. Aim 3 will clarify the role of the large, newly discovered cellular elements in initial biofilm development and in antibiotic susceptibility. Aim 4 will characterize the vegetations within discarded in vivo vascular catheters removed from postsurgical, trauma, and critically ill patients, and compare these data with biofilm vegetations cultivated in vitro. Experimental methods include standard microbiological methods, confocal microscopy and high resolution electron microscopy, cell sorting, fluorescence in situ hybridization, and biochemical analyses. Resulting data will significantly contribute to our understanding of the initiation and treatment of biofilm infections, especially those associated with postsurgical, trauma, and critically ill patients.